The following description provides a summary of information relevant to the present invention and is not a concession that any of the information provided or publications referenced herein is prior art to the presently claimed invention.
1. Area of the Art
The invention relates generally to fluorescence sensor molecules and specifically to a new group of modular fluorescence sensor molecules.
2. Description of the Prior Art
Numerous assay methods have been developed for the detection and quantitative determination of analytes contained in biochemical samples. A substantial portion of the currently used assay methods relies on specific binding reactions between analytes and assay reagents. The analytes may be large complex molecules, such as proteins, viruses, viral antigens, bacterial cells, cell surface receptors, enzymes, hormones, polysaccharides, glycoproteins, lipoproteins, or small haptenic molecules, such as peptides, certain hormones, therapeutic drugs, and drugs of abuse, to name a few.
The binding assays can be divided into two major groups based on their format: homogeneous and heterogeneous assays. Homogeneous assays are based on a single-phase reaction between analyte and assay reagents. Heterogeneous assays, typically, involve binding of an analyte contained in the liquid sample to assay reagents, which are attached to a solid support. Various materials have been used as support surfaces, including glass rods, glass beads, silica impregnated strips, glass fiber, and microparticles.
Dyes in general, and fluorescent dyes in particular, are commonly utilized in both homogeneous and heterogeneous binding assays to provide a detectable signal. However, an accurate detection of fluorescent signals produced by analytes bound to the labels is often hindered by a high and variable background due to the fluorescence of the biological sample itself.
Liquid flow cytometry helps to overcome this problem. In flow cytometry, labeled particles with bound analyte are passed through a laser beam. The emitted fluorescent signals of the particles are measured and correlated to the presence and quantity of the analyte. The main advantage of this method is its capability of accurate detection and measurement of the fluorescent signals associated with the bound analyte in the presence of other unbound constituents of the sample.
Saccharides represent an important group of biochemical analytes. Current methods for determining their concentrations in a sample typically rely on enzymatic assays. Although enzymatic assays have proven to be reliable, they must utilize rather unstable enzymes, which become exhausted in the presence of their substrates. Additionally, conventional enzymatic assay methods cannot be utilized in a convenient flow cytometry format. Particle-based assays, such as the ones used in flow cytometry, require a signal change confined to the particle. Normal enzymatic analysis methods use freely diffusable intermediates that violate this requirement.
Determination of saccharides is particularly important in clinical settings. Treatment of diabetes and hypoglycemia requires frequent measurement of tissue glucose concentration. This is commonly accomplished by drawing a small blood sample (as by a fingerstick) several times daily. A patient typically uses a lancet to draw a droplet of blood and applies the droplet to a reagent strip which is read in a small meter. This process is painful, invasive, and time-consuming.
Recently, a minimally invasive method for measuring glucose in vivo has been disclosed in U.S. patent application Ser. No. 09/393,738 filed on Sep. 10, 1999, which has been commonly assigned to the assignee of the present invention and is incorporated by reference herein. The method is based on the use of implanted sensor particles capable of generating a detectable analyte signal in response to the analyte concentration in the body. The proposed method is less intrusive than the conventional fingerstick technique for measuring blood D-glucose. It requires only periodical replacement of the sensor particles in the skin.
The sensor particles typically comprise fluorescence sensors either attached to the surface or incorporated into the body of the particles. The sensors are specific to the target analyte. The binding of the sensor to the analyte generates a detectable signal that is responsive to the concentration of the analyte. When the analyte is glucose, diboronic acids conjugated to fluors are used.
Similar fluorescent sensors, which are specific to glucose, are also described by James et al., in the Journal of the American Chemical Society, 1995, vol. 117 pp. 8982-8987, by James et al. in U.S. Pat. No. 5,503,770, and by Takeuchi et al. in Tetrahedron, vol. 52, No. 4, pp. 1195-1204. Briefly, the fluorescent sensors of James and Takeuchi have the following general formula:

In the formula, F designates a fluorophore, R is a lower aliphatic or aromatic group, and n+m is 2 or 3. The fluorescent intensity of the sensor changes in response to photo-induced electron transfer (PET) between the amine group and the fluorophore as modulated by binding of glucose hydroxyls to a pair of boronic acids. In the absence of glucose binding, the fluorescence by the fluorescent group is quenched by the unshared electron pair of the nitrogen atom. When glucose is bound, the unshared electron pair is utilized in the bond formation and does not participate in fluorescence-quenching. Consequently, intrinsic fluorescence of the sensor is expressed.